1. Field of the Invention
The present invention in a general and overall sense relates to the field of recombinant proteins and peptides. The invention also relates to the field of molecular processes and methods for producing recombinant proteins particularly methods that employ E. coli as an expression vehicle. The invention also relates to compositions and methods for preparing pro-insulin, insulin, and both of these alone or in combination with each other and with other compositions.
2. Background of the Related Art
Insulin is a polypeptide hormone secreted by beta-cells of the pancreas. This hormone is made up of two polypeptide chains, an A-chain of 21 amino acids, and a B-chain of 30 amino acids. These two chains are linked to one another in the mature form of the hormone by two interchain disulfide bridges. The A-chain also features one intra-chain disulfide bridge.
Insulin is a hormone that is synthesized in the body in the form of a single-chain precursor molecule, pro-insulin. Pro-insulin is a molecule comprised of a prepeptide of 24 amino acids, followed by the B-chain peptide, a C-peptide of 35 amino acids, and an A-chain peptide. The C-peptide of this precursor insulin molecule contains the two amino acids, lysine-arginine (LR) at its carboxy end (where it attaches to the A-chain), and the two amino acids, arginine-arginine (RR) at its amino end (where it attaches to the B-chain). In the mature insulin molecule, the C-peptide is cleaved away from the peptide so as to leave the A-chain and the B-chain connected directly to one another in its active form.
Molecular biology techniques have been used to produce human pro-insulin. In this regard, three major methods have been used for the production of this molecule. Two of these methods involve Escherichia coli, with either the expression of a large fusion protein in the cytoplasm (Chance et al. (1981), and Frank et al (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich, D. and Gross, E., eds.), pp. 721-728, 729-739, respectively, Pierce Chemical Company, Rockford, Ill.), or the use of a signal peptide to enable secretion into the periplasmic space (Chan et al (1981) P.N.A.S., U.S.A., 78:5401-5404). A third method utilizes yeast, especially Saccharomyces cerevisiae, to secrete the insulin precursor into the medium (Thim, et al. (1986), P.N.A.S., U.S.A., 83:6766-6770).
Chance et al. report a process for preparing insulin by producing each of the A and B chains of insulin in the form of a fusion protein by culturing E. coli that carries a vector compromising a DNA encoding the fusion protein, cleaving the fusion protein with cyanogen bromide to obtain the A and the B chains, sulfonating the A and B chains to obtain sulfonated chains, reacting the sulfonated B chain with an excess amount of the sulfonated A chain; and then purifying the resultant products to obtain insulin. Drawbacks associated with this process are that it requires two fermentation processes and the requirement of a reaction step for preparing the sulfonated A chain and the sulfonated B chain. This results in a low insulin yield.
Frank et al. described a process for preparing insulin in the form of a fusion protein in E. coli. In this process, pro-insulin is produced in the form of a fusion protein by culturing E. coli which carries a vector comprising a nucleic acid sequence (DNA) encoding for the fusion protein, cutting the fusion protein with cyanogens bromide to obtain pro-insulin, sulfonating the pro-insulin and separation of the sulfonated pro-insulin, refolding the sulfonated pro-insulin to form correct disulfide bonds, treating the refolded pro-insulin with trypsin and carboxypeptidase B, and then purifying the resultant product to obtain insulin. However, the yield of the refolded pro-insulin having correctly folded disulfide bonds is reported to sharply decrease as the concentration of the pro-insulin increases. This is allegedly due to, at least among other reasons, to misfolding of the protein, and some degree of polymerization being involved. Hence, the process entails the inconvenience of using laborious purification steps during the recovery of pro-insulin.
Thim et al. report a process for producing insulin in yeast, Sacchromyces cerevisiae. This process has the steps of producing a single chain insulin analog having a certain amino acid sequence by culturing Sacromyces cerevisiae cells, and isolating insulin there from through the steps of: purification, enzyme reaction, acid hydrolysis and a second purification. This process, however, results in an unacceptably low yield of insulin.
The role of the native C-peptide in the folding of pro-insulin is not precisely known. The dibasic terminal amino acid sequence at both ends of the C-peptide sequence has been considered necessary to preserve the proper processing and/or folding of the pro-insulin molecule to insulin.
Other amino acids within the within the C-peptide sequence, however, have been modified with some success. For example, Chang et al. (1998) (Biochem. J., 329:631-635) described a shortened C-peptide of a five (5) amino acid length, —YPGDV—(SEQ ID NO: 1), that includes a preserved terminal di˜basic amino acid sequence, RR at one terminal end, and LR at the other terminal end, of the peptide. Preservation of the dibasic amino acid residues at the B-chain-C peptide (B-C) and C-peptide-A-chain junctures is taught as being a minimal requirement for retaining the capacity for converting the pro-insulin molecule into a properly folded mature insulin protein. The production of the recombinant human insulin was described using E. coli with a shortened C-peptide having a dibasic amino acid terminal sequence.
One of the difficulties and/or inefficiencies associated with the production of recombinant insulin employing a pro-insulin construct having the conserved, terminal di-basic amino acid sequence in the C-peptide region is the presence of impurities, such as Arg-insulin, in the reaction mixture, once enzymatic cleavage to remove the C-peptide is performed. This occurs as a result of misdirected cleavage of the pro-insulin molecule so as to cleave the C-peptide sequence away from the A-chain at this juncture, by the action of trypsin. Trypsin is a typical serine protease, and hydrolyses a protein or peptide at the carboxyl terminal of an arginine or lysine residue (Enzymes, pp. 261-262 (1979), ed. Dixon, M. & Webb, E. C. Longman Group Ltd., London). This unwanted hydrolysis results in the unwanted ARG-AO-insulin by-product, and typically constitutes about 10% of the reaction yield. Hence, an additional purification step is required. The necessity of an additional purification step makes the process much more time consuming, and thus expensive, to use. Moreover, an additional loss of yield may be expected from the necessity of this additional purification step.
Others have described the use of pro-insulin constructs that do not have a conserved terminal dibasic amino acid sequence of the C-peptide region. For example, U.S. Pat. No. 6,777,207 (Kjeldsen et al.) relates to a novel pro-insulin peptide construct containing a shortened C-peptide that includes the two terminal amino acids, glycine-arginine or glycine-lysine at the carboxyl terminal end that connects to the A-chain of the peptide. The B-chain of the pro-insulin construct described therein has a length of 29 amino acids, in contrast to the native 30 amino acid length of the native B-chain in human insulin. The potential effects of this change to the native amino acid sequence of the B-chain in the human insulin produced are yet unknown. Methods of producing insulin using these pro-insulin constructs in yeast are also described. Inefficiencies associated with correct folding of the mature insulin molecule when yeast utilized as the expression host, render this process, among other things, inefficient and more expensive and time consuming to use. In addition, yeast provides a relatively low insulin yield, clue to the intrinsically low expression levels of a yeast system as compared to E. coli. 
As evidenced from the above review, a present need exists for a more efficient process for production of human insulin that is efficient eliminates currently necessary purification steps, and that at the same time improves and/or preserves acceptable production yield requirements of the pharmaceutical industry.
The above references are incorporated by reference herein where appropriate for appropriate teachings of additional or alternative details, features and/or technical background.